This project will uncover the roles of Sox4, Sox11, and Sox12 in skeletogenic cells in vivo. These three highly related proteins form the subfamily C of Sry-related HMG box (Sox) transcription factors. Their genes are co-expressed in skeletogenic mesenchymal cells throughout the mouse embryo, and remain differentially expressed when these cells undergo chondrocyte and osteoblast differentiation. While the roles of Sox12 in vivo remain unknown, mice lacking Sox4 and/or Sox11 have started to reveal important roles for these two genes in multiple steps of skeletogenesis. The molecular roles of Sox C proteins remain largely unknown, but preliminary studies have suggested that they may bind DNA and activate transcription similarly but with different efficiencies. These data raise the hypothesis that Sox C proteins may have redundant, complementary, or distinct molecular roles in controlling skeletogenic cell fate and differentiation. To test this hypothesis, Aim 1 is to further study the roles of the Sox C proteins in mouse embryo skeletogenesis. Mice carrying Sox11 and Sox12 conditional null alleles will be generated using the same strategy that was used to generate a Sox4 conditional null allele. Well-established Cre transgenes will be used to specifically inactivate the genes in the entire embryo or at specific steps during skeletogenesis. Skeletal defects will be characterized at the morphological, cellular and molecular levels, and primary cell culture experiments will be carried out to complement in vivo studies. Aim 2 is to characterize the molecular roles of Sox C proteins in skeletogenic cells. The DNA-binding and transactivation properties of the three Sox C proteins will be further studied using standard in vitro and cell culture assays. Potential target genes will be identified by gene expression array screening. The action of Sox C proteins on target gene regulatory sequences will be studied using DNA-binding and transactivation assays in vitro and in vivo. It is anticipated that this study will identify Sox C proteins as essential determinants of skeletogenic cells from pluripotent precursor stages until late maturation in specific cell lineages, and will thereby provide significant insights into molecular mechanisms involved in normal skeletogenesis and in genetic and acquired diseases of the skeleton.